[Title]

Quantitation and composition of cutaneous microbiota in diabetic and nondiabetic men

[Author's name (all)]
Henry Redel, Zhang Gao, Alexander V. Alekseyenko, Yanjiao Zhou, Guillermo I. Perez-Perez, George
Weinstock, Erica Sodergren, and Martin J. Blaser.
[Journal name, Volume (Issue), page, year published]
The journal of infection diseases (2013); 207:1105-14
[About the journal, Impact factor]
Published continuously since 1904, The Journal of Infectious Diseases (JID) is the premier global journal
for original research on infectious diseases. The editors welcome Major Articles and Brief Reports
describing research results on microbiology, immunology, epidemiology, and related disciplines, on the
pathogenesis, diagnosis, and treatment of infectious diseases; on the microbes that cause them; and on
disorders of host immune responses. JID is an official publication of the Infectious Diseases Society of
America. Published 12 times per year.
Impact factor 5.997
[Type of article]
Original Article
[Background of the first author (affiliation, occupation)]
Department of Medicine, New York University School of Medicine
[Background of the group authors]
Zhan Gao from Department of Medicine, New York University School of Medicine.
Huilin Li from Department of Environmental Medicine, New York University School of Medicine.
Alexander V. Alekseyenko from Department of Medicine and Center Health Informatics and
Bioinformatics, New York University School of Medicine.
Yanjio Zhou and George Weinstock from The Genome Institute, Department of Genetics, Washington
University School of Medicine, St. Louis, Missouri.
Guillermo I. Perez-Perez and Martin J. Blaser from Department of Medicine and Department of
Microbilogy, New York University School of Medicine.
[Kind of research topic has the authors research group been investigated]
The authors studied examine microbiota of diabetic skin prior to ulcer development or infection.
[Basic knowledge necessary to understand the literature]
Bacteria Phyla
Major group bacteria phyla: Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidates.

[Research] background
What research problem that a researcher would like to answer?
Data on the composition of the cutaneous microbiota of diabetic patients is lacking.
What is already known about the topic?
- By using the culture, infection are polymicrobial, with Pseudomonas aeruginosa, Escherichia coli and
Staphylococcus species being the most prevalent isolates.
- Staphylococcus species account for 70% of the normal skin microbiome on the plantar aspect of the
foot.
- Only 7% of the microbiota was presented by Staphylococcus species on the opposite limb skin of
diabetic foot ulcer patients.
What is already unknown about the topic?
Differences skin organism on the feet in people leaving with diabetes mellitus and non diabetes mellitus.
[Aim or Objectives]
The aim of this study was to determine whether there are differences in surface microbiota of the arm and
foot between nondiabetic men and diabetic men without a history of diabetic foot ulcer.
[Hypothesis]
The bacterial community structure and quantitate of the foot in diabetic and control (nondiabetic) is
similar.
[Methods]
Observational study (Case-control)
[Variables (Independent variable and dependent variable)]
Independent variables: Quantitation and composition of cutaneous microbiota
Dependent variable: Diabetic and nondiabetic men
[Controlling external factors and internal factors]
Exclusion criteria included any history of a diabetic foot ulcer, a body mass index (BMI; defined as the
weight in kilograms divided by the height in square meters) of > 40, active dermatologic conditions at
sampled anatomical sites, corticosteroid or antibiotic use within the prior month, known active infection
at any site, and inability to take daily showers.
[Sampling design]
Seventy men aged 1870 years, with or without diabetes, were enrolled. The subjects were recruited from
the outpatient clinic of the VANYHHS (Manhattan campus). Enrollment was stopped when we obtained
30 evaluable subjects in the diabetic group and 30 in the nondiabetic (i.e., control) group.

[Analytical methods and collection of data]

Data collection
Skin Sampling
Samples were collected with cotton swabs (Fisher Scientific) soaked in 0.15 M NaCl and 0.1% Tween 20
(Fisher Scientific).
Site swabbed: the right forearm and the dorsum, plantar arch, and first interdigital space of the right foot
(30x swabbed back and forth) and the procedure were repeated with a second swab.
Specimen Processing
Specimens were obtained under sterile conditions, placed in 1.5-mL Eppendorf tubes, and stored at
80C. The samples were thawed for extraction, performed with the MoBio PowerLyzer Power Soil
DNA Isolation Kit (MoBio Laboratories), according to the manufacturers protocol. Samples were
divided into aliquots for qPCR and 454 pyrosequencing.
Analysis
Wilcoxon/ Student t-tests and ANOVA analyzed using SAS 9.2.
[Results]
Table 1: Demographic and Clinical Characteristics of 30 Subjects Without Diabetes (Control) and 30
Subjects With Diabetes
Baseline characteristics were generally similar for the 2 groups. As expected, the diabetic group had a
significantly higher BMI and rate of statin use (P < .05) and higher levels of hemoglobin A1c and serum
glucose.
Figure 1: Quantitation of bacteria from four sites.
Figure 1A
- The total number bacterial amplicons higher in the interdigital space than for all other studied sites.
- Total bacterial counts also were higher on the plantar aspect of the foot, compared with the dorsal
aspect of the foot and the forearm.
Figure 1B
The number of Staphylococcus copies present in the control group at the first interdigital space of the foot
was greater in the control group, compared with the diabetes group.
Figure 1C
Diabetic men had an increased number of S. aureus at the plantar aspect of the foot and at all sites
combined, compared with controls.
Figure 1D
Total fungal counts were higher in the interdigital space and on the forearms than on the plantar and
dorsal aspects of the foot.
Figure 2: Rate of Staphylococcus aureus carriage.
- The rate of S. aureus carriage at each of the 4 sites did not significantly differ between the control
and the diabetic groups.
- The combined S. aureus carriage rate on the forearm, including both controls and diabetic subjects,
was higher than the carriage rate at the interdigital space (28% vs 8%; P = .008).

Table 2: Representation of Staphylococcus aureus in Relation to Other Bacterial Taxa, by Body Site

The plantar aspect of the foot, more men in the diabetic group than the control group had S. aureus
detected at levels >0.01% of the total bacterial population.
Figure 3: Alpha diversity rarefaction plots by site and diabetes status
- The plantar aspect of the feet of diabetic subjects had greater taxonomic richness and Shannon
diversity at all taxonomic levels, com- pared with the plantar aspect of the feet of controls.
- Forearm samples from both controls and diabetic men had similar alpha diversity to each other and to
the diabetic foot samples.
Figure 4: Composition of microbial communities
- Among arm samples, no significant differences in relative abundances were found between diabetic
and nondiabetic subjects.
- Among foot specimens, the relative abundance of Firmicutes was greater in controls, compared with
diabetic subjects.
- Actinobacteria had a greater relative abundance in foot specimens from diabetic men, compared with
foot specimens from controls.
- At the genus level, Staphylococcus had the greatest relative abundance in foot specimens from both
controls and diabetic patients. Contritely, Corynebacterium abundance was greater in the feet of
diabetic subjects (18.2%) than in the feet of controls.
Figure 5: Principal Coordinate Analysis (PCoA) of weighted Unifrac beta-diversity
Figure 5A
There were no significant differences in the composition of the microbiota at the forearm.
Figure 5B and 5D
Foot communities split according to diabetes status
Figure 5C
The intergroup beta-diversity in the arm samples had intermediate values, suggesting that diabetic
samples form a subcluster with the controls, which was evident from the PCoA plot.
Figure 5E
There was increased intragroup beta-diversity in foot samples of diabetic men.
Figure 5F
In arm specimens, the trend was reversed; the arms of diabetic men had lower intragroup beta-diversity
than the controls.
[Discussion]
New finding in this research
The bacterial populations at the plantar aspect of the feet of diabetic men were more diverse and had
different composition, compared with values for nondiabetic men.
Interpretations of the results
- The quantity of Staphylococcus organisms was reduced on the diabetic plantar aspect of the foot,
whereas the more virulent S. aureus was present in greater quantities; these changes may be
precursors to diabetic foot infection and ulcer development.
- The diabetic foot has a lower population of Staphylococcus species present.

Implications to clinical practice

Three dimensions of bioburden may be important in the nonhealing DFU: microbial load, microbial
diversity and/or pathogenicity.1 Thus, this study strengthened the role of bacteria diversity in diabetes
men.
From this study, we comprehend that the bacterial population at the plantar feet of diabetic men more
diverse and increases of S. aureus as pathogenic. This is suggesting for diabetic men should more be
careful to maintaining foot hygiene.

Limitation of the study

The sampling occurred over a number of months, and there was a trend toward more cases being recruited
during warmer months of the study.
[Critique]
-

In this study shown the characteristic of BMI was different, it might be influence of bacteria diversity
on the skin. Previous study revealed, the significant positive correlation between inflammatory status
(TNF-) on the skin and BMI2, and the inflammatory status affected diversity of bacteria especially
for Staphylococcus aureus.3

This study only interpreted bacteria diversity in diabetes men, therefore the future study is necessary
to comprehend bacteria status in diabetes women.

Reference:
1. Gardner SE, Frantz RA. Wound bioburden and infected-related complications in diabetic foot
ulcer. Biol Res Nurs 2008; 10:44-53.
2. Ogai K, Matsumoto M, Aoki M, Minematsu T, Kitamura K, et al. Increased level of
tumor factor-alpha (TNF-) on the skin of Japanese obese males: measured by quantitative
skin blotting. International Journal Cosmetic Science 2016; 1-8.
3. Sayedyahossein S, Xu SX, Rudkouskaya A, McGavin MJ, et al. Staphylococcus
aureus keratinocyte invasion is mediated by integrin-linked kinase and Racl.
The FASEB Journal 2015; 29(5): 711-723.